Heart disease is the leading cause of death worldwide, but the genetic risk factors for congenital heart defects and adult cardiac disorders are incompletely understood. Variation in distant-acting regulatory sequences (enhancers) is likely to contribute significantly to heart development and disease via regulation of gene expression. However, the genomic location and in vivo function of heart enhancers in the human genome remains largely unknown, hindering efforts to establish mechanistic links between enhancers and heart development and clinical cardiac phenotypes. Our laboratories have pioneered techniques for genome-wide enhancer identification by ChIP-seq and in vivo functional enhancer characterization via transgenic mouse experiments. Here, these techniques will be leveraged to address the pressing need for accurate annotation of heart enhancer location in the human genome and activity across heart developmental and disease states. Despite the general value of mouse models and in vitro studies of human cell lines, we have shown that there is significant divergence in sequence and functional conservation between human and mouse heart enhancers and that enhancer maps generated from specific cell lines capture a limited slice of the regulatory elements active in heterogeneous tissues such as the heart. The present proposal is aimed at characterizing heart enhancers and associated gene expression directly from human tissues representing different developmental stages and subregions of the fetal and adult heart, with the goal of generating datasets of significant value to basic and clinical cardiac research. We additionally propose to examine inter-individual variation in heart enhancer activity and gene expression, characterizing differences across healthy subjects and individuals with heart disease. In proof-of-principle studies, we have demonstrated the general feasibility and scientific impact of this approach. The specific aims of this proposal include: 1) we will perform ChIP-seq targeting enhancer- associated epigenomic marks to identify in vivo enhancers directly from comparisons of human heart tissues, including pre- and postnatal developmental stages, cardiac subregions, and major adult cardiac disease states. We will also perform transcriptome profiling by RNA-seq in the same samples. 2) We will perform at least 250 transgenic mouse assays to characterize in vivo activity patterns of candidate developmental and disease-relevant heart enhancers and study functional effects of putative deleterious non-coding sequence variation. Tested loci will be selected based on biomedical interest and will include community-nominated cardiac loci and risk variants. 3) We will present the results as an integrated community resource for heart genetics, providing a web portal for data browsing and download and molecular reagents and transgenic mice to enable downstream studies of regulatory sequences in heart development and disease. This research will reveal the regulatory landscape of human cardiac development, function, and disease, and will address the pressing national need for genomic resources to enable and accelerate the advancement of cardiac research. (End of Abstract)